PROJECT ABSTRACT Cancer metastasis is the most deadly aspect of cancer, responsible for 90% of cancer-related deaths. A key aspect of the metastatic cascade involves cancer cell migration through the stroma microenvironment around the primary tumor. Macrophages in the stroma have been found to play a critical role in cancer progression and regulation of cancer cell migration. Broadly speaking, activated (polarized) macrophages can exhibit an anti-tumor (M1) or pro-tumor (M2) phenotype. Polarized macrophages display remarkable plasticity in response to the tumor milieu and extracellular matrix (ECM) properties. Increased ECM stiffness as well as altered viscoelasticity is a feature of various cancers. The impact of soluble cues and ECM stiffness on macrophage polarization has been studied. However, the impact of alterations in ECM viscoelasticity on macrophage polarization, migration, and signaling to cancer cell has not been investigated. A consequence of viscoelasticity is a reduction in resistance to deformation over time (stress relaxation). The overall hypothesis of the current work is that faster matrix stress relaxation preferentially polarizes macrophages to an M2-phenotype, promotes macrophage migration, and facilitates cancer migration in 3D co- culture. To address this hypothesis, the proposed work is divided into three aims. The first aim is to develop alginate-collagen based matrices that mimic the stroma microenvironment, in which stiffness and stress relaxation can be independently modulated independent of collagen fiber architecture. Preliminary results indicate that varying alginate molecular weight, polymer concentration, and polymerization temperature in the collagen-alginate matrices enables these tunabilities. The second aim is to determine how matrix stress relaxation influences macrophage polarization and migration. Preliminary results demonstrate the feasibility of 3D encapsulation and quantification of migration parameters. The third aim is to uncover the impact of matrix stress relaxation on interactions between macrophages and cancer cells, and determine the underlying mechanisms. The proposed work is significant because it will reveal how matrix viscoelasticity, a key characteristic of the tumor microenvironment, regulates macrophage polarization and phenotype. This is innovative because while there have been previous studies of the impact of stiffness on macrophages, this will be the first study to elucidate how matrix stress relaxation mediates macrophage migration, phenotype, and signaling to cancer cells. The development of materials is also innovative because it aims to develop a stroma- mimic matrix with tunable stiffness and stress relaxation over a broader range than has been previously shown. Successful completion of the proposed work will provide new insight for macrophage-centered approaches to preventing metastasis. This is relevant to the part of NIH's mission that seeks to develop fundamental knowledge that will help reduce the burdens of human disability and disease.